PROJECT SUMMARY Chromatin structure and gene expression are controlled by histone post-translational modifications (PTMs) on nucleosomes, the basic repeating unit of chromatin. Chromatin decondensation is controlled in part by the PAD4-dependent conversion of arginine to citrulline on histones (i.e. citrullination). Significantly, PAD4 activation in neutrophils initiates a cell death program distinct from apoptosis or necrosis, wherein chromatin is hypercitrullinated, decondensed, and extruded from the cell in neutrophil extracellular traps (i.e. NETosis). The release of citrullinated chromatin into the blood is thought to contribute directly to PAD4-related pathologies. Thus, citrullinated nucleosomes (Cit-Nucs) are a promising blood-accessible biomarker for a range of autoimmune diseases, including rheumatoid arthritis (RA), cancer thrombosis, and sepsis. However, there are no assays capable of reliably quantifying nucleosome citrullination from serum or plasma. Current assays that aim to quantify Cit-Nucs use citrullinated histones as standards for assay development and calibration. These type of standards are problematic for two major reasons; First, citrullinated histones fail to provide accurate quantification of nucleosomes, especially at low concentrations; second, free histones are highly charged and readily aggregate in plasma, which significantly impacts their linear recovery from plasma samples. By contrast, we (and others) have found that nucleosomes are highly stable in plasma, suggesting that these substrates may provide superior standards for assay quantification. EpiCypher is pioneering the commercialization of recombinant designer nucleosomes (dNucs) carrying physiological histone PTMs for next-generation epigenetics assays. Here, we are developing CitNuc?, the first ELISA to accurately quantify Cit-Nucs for NETosis research and preclinical biomarker development. Our innovative assay uses recombinant citrullinated designer nucleosomes (Cit-dNucs) as quantification standards for antibody pair selection / validation and reliable assay quantification. Importantly, unlike histones, Cit-dNucs can be faithfully recovered from plasma samples, enabling development of a highly sensitive ELISA. In Phase I, we successfully used Cit-dNucs to identify highly specific antibodies and establish reliable standards for quantification of Cit-Nucs in plasma. We also examined key bioanalytical parameters and validated the ability of CitNuc ELISA to detect differences in Cit-Nucs between healthy and RA patient samples, demonstrating the utility of our assay in a clinical environment. In Phase II, we will complete bioanalytical testing and define reliable lot-release strategies for CitNuc ELISA kits (Aim 1), paving the way for commercialization. In Aims 2 and 3, we will develop the preclinical application of this assay for biomarker discovery, focusing on RA and cancer thrombosis, two diseases associated with high levels of PAD4-dependent nucleosome citrullination. Together, these Aims will result in the commercial launch of a first-generation CitNuc ELISA assay, which will be marketed for NETosis research and biomarker development applications.